Amino acids are used in human medicine, in the pharmaceutical industry, in the foodstuffs industry and, very particularly, in animal nutrition.
It is known that amino acids are prepared by fermenting strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of their great importance, efforts are continuously being made to improve the preparation methods. Methodological improvements can concern measures relating to fermentation, such as stirring and supplying with oxygen, or the composition of the nutrient media, such as the sugar concentration during the fermentation, or the workup to the product form by means of ion exchange chromatography, for example, or the intrinsic performance properties of the microorganism itself.
Methods of mutagenesis, selection and mutant choice are used for improving the performance properties of these microorganisms. This results in strains which are resistant to antimetabolites or auxotrophic for metabolites of regulatory importance and which produce amino acids. A known antimetabolite is the lysine analog S-(2-aminoethyl)-L-cysteine (AEC).
For some years now, methods of recombinant DNA technology have also been used for improving L-amino acid-producing strains of corynebacterium by amplifying individual amino acid biosynthesis genes and investigating the consequences of this for amino acid production. A review of a wide variety of aspects of the genetics, the metabolism and the biotechnology of Corynebacterium glutamicum can be found in Pühler (chief ed.), Journal of Biotechnology 104 (1-3), 1-338, 2003.
The nucleotide sequence of the Corynebacterium glutamicum malate quinone oxidoreductase-encoding gene was determined by Molenaar et al. (European Journal of Biochemistry 254: 395-403 (1998)) and is available to the public in the database of the National Center for Biotechnology Information (NCBI) at the National Library of Medicine (Bethesda, Md., USA) under the accession number AJ224946.
It is furthermore to be found in patent application WO 01/00844 as sequence No. 569 and sequence No. 571 as well as in patent application EP-A-1108790 as sequence No. 3478, sequence No. 7065 and sequence No. 7066.
EP1038969 describes an improvement in the fermentative production of L-amino acids by coryneform bacteria which results from amplifying the mqo gene.
On the other hand, WO 02086137 describes the beneficial effect on L-amino acid production by coryneform bacteria which is achieved by attenuating the mqo gene. A mutation of the mqo gene which is described in the application and which is designated “allele 672” carries the nucleotide adenine in place of the nucleotide guanine at position 672 in the DNA sequence of the mqo gene, with this resulting in the formation of a stop codon at position 224 in the amino acid sequence of the Corynebacterium glutamicum malate quinone oxidoreductase. The “allele 1230”, which, in addition to the mutation in allele 672, also contains a cytosine to thymine transition at position 1230 in the nucleotide sequence of the mqo gene, is also described. The application furthermore describes the elimination of the mqo gene as a result of gene interruption brought about by integration mutagenesis, with this leading to an increase in the production of L-lysine by the corresponding strain.
The microbial biosynthesis of L-amino acids in coryneform bacteria is a system which is complex and multilayered, being interlinked with a variety of other metabolic pathways in the cell. It is therefore not possible to make any prediction as to whether complete elimination of, or a reduction in, the catalytic activity of the malate quinone oxidoreductase will improve the production of L-amino acids at different steps. It is therefore desirable to also have available malate quinone oxidoreductase variants which differ in the degree of their activity.
For the sake of greater clarity, the nucleotide sequence of the Corynebacterium glutamicum malate quinone oxidoreductase-encoding mqo gene (wild-type gene) in accordance with the information supplied by the NCBI database is depicted in SEQ ID NO: 1 while the ensuing amino acid sequence of the encoded malate quinone oxidoreductase is depicted in SEQ ID NO: 2 and 4. Nucleotide sequences which are located upstream and downstream are also given in SEQ ID NO: 3.